Biomolecular condensates are membraneless organelles that form through liquid-liquid phase separation of proteins and nucleic acids. They are essential for organizing biochemical reactions in cells, and their assembly and disassembly are often regulated through enzymatic activities such as phosphorylation or dephosphorylation. Despite extensive studies on phase separation, how enzymatic activity governs the kinetics and internal structure of condensates remains poorly understood, partly due to the challenge of probing relevant time and length scales. This thesis provides a state-of-the-art overview of biomolecular condensates and the fundamental principles of small-angle scattering techniques used to study their structure and dynamics across multiple scales. In this thesis, we investigate condensates formed by an arginine-rich phosphorylatable peptide (RRASLRRASL) and poly (uridylic acid) (polyU) RNA as a simple model system. All experiments were carried out at 37°C to maintain physiological conditions. Using time-resolved ultra-small angle X-ray scattering (TR-USAXS), small angle neutron scattering (SANS), confocal fluorescence microscopy, spectrophotometry and coarse-grained molecular dynamics simulations, we capture the mechanisms of condensate assembly and disassembly over time scales ranging from milliseconds to minutes and across nanometer to micrometer length scales. Under passive conditions, where condensates form spontaneously through electrostatic interactions between the peptide and RNA, we determined the phase diagram and critical concentrations governing phase separation. Absorbance and confocal imaging revealed micrometer-sized spherical droplets that reach steady-state within minutes. TR-USAXS experiments with synchrotron source show that the mean condensate radius increased with time following a t^1/3 scaling law, consistent with growth driven by coalescence or Ostwald ripening, while the number density decayed as t^-1. Complementary coarse-grained molecular dynamics simulations show that peptide-decorated RNA chains rapidly assemble after mixing, forming the subunits of condensate growth. Under active conditions, condensate formation is enzymatically triggered by the dephosphorylation of initial phosphorylated peptide by Lambda Protein Phosphatase (LPP). Spectrophotometry and TR-USAXS measurements revealed delayed condensate formation and a two-step growth mechanism: an initial formation of loosely packed, fractal-like clusters followed by coalescence into denser spherical droplets. Fluorescence recovery after photobleaching (FRAP) indicated higher peptide diffusivity inside actively formed droplets, consistent with their loosely-packed local structure. Protein Kinase A (PKA) is used to study the enzymatic disassembly of preformed condensates. Phosphorylation of serine residues by PKA neutralized the peptide's positive charge, weakening its electrostatic attraction with RNA, resulting in condensate dissociation. Using TR-USAXS and numerical simulations, we observed the gradual dissociation of condensates over time, highlighting how enzymatic activity can actively reverse phase separation. Overall, my PhD research combines advanced scattering techniques, real-time confocal microscopy, and molecular simulations to uncover the fundamental principles of condensate formation and to demonstrate how enzymatic activities regulate their assembly, growth, and disassembly. It provides an integrated view of how biochemical processes govern the structure and dynamics of membraneless organelles in living cells.
Tamizhmalar Sundararajan (Thu,) studied this question.